1. Field of the Invention
The present invention relates to a method for making uniform polymeric microbeads of predetermined size and further relates to polymeric microbeads which have an attached detectable label.
2. Description of the Prior Art
Flow cytometry is the preferred method for the rapid detection and measurement of a variety of cellular constituents. In this technique, measurement of large numbers of individual cells in monodisperse fluid suspension is done by electrical and/or optical techniques. Physical sorting of selected cell subpopulations may also be performed. Fluorescence activated flow cytometry depends on measuring the fluorescent emission of each cell, and provides rapid and accurate statistical data at rates of up to 5000 cells per second, or perhaps higher. Cells which exhibit native fluorescence can be used, or fluorescence can be induced by binding a suitable fluorescent label to the cell structure being analyzed.
Flow cytometers are used to differentiate, count and separate subpopulation of cells by either size, fluorescence or other useful parameters. In general, cells in suspension are introduced to the center of a liquid stream and caused to pass, substantially one at a time, through the focused beam from a light source. In passing through the light beam, the cell, if labeled with a fluorescent marker, is caused to emit one or more fluorescent signals. Analysis of the intensity, color or polarization of the fluorescent signal allows individual characterization of the cell.
There is a need for a method to calibrate a fluorescence activated flow cytometer by means of a standard cell having a known quantity of fluorescent molecules per cell. The principal requirement for a standard cell is stability and long shelf time. Of course, due to the transient lifetime of living cells, it is impossible to prepare suitable standard cells from living cells. Polymeric particles having the same size (average 4 to 10 microns) and conformation as living cells could be used as flow cytometry standards if they could be suitably labeled.
Polymeric microparticles in general have been prepared by the conventional technique of emulsion polymerization. By this technique, microparticles with a high degree of size uniformity can be obtained, but these particles have been limited to about 2 microns or less in diameter. Larger particles have been obtained by a variety of procedures from pre-formed polymers, however, these techniques all give particles of wide variability in size, shape and other physical properties.
U.S. Pat. No. 4,336,173 to Ugelstad discloses a method for preparing uniform microbeads of larger size than those available by emulsion polymerization. In the Ugelstad method, a dispersion of microparticles of small, but uniform, size produced by emulsion polymerization is contacted with a first substance which is absorbed into the particles to cause some swelling of the particles. Subsequently, a second substance, usually a polymerizable monomer, is likewise absorbed into the particles. Upon internal polymerization of this monomer, the particle undergoes additional swelling. The desired particle size is reached by absorbing pre-calculated amounts of the first and second substances into the microparticle.
Preparation of microbeads in which a dye is incorporated is known. In U.S. Pat. No. 4,194,877 to Peterson, polymeric microbeads are disclosed in which a dye is emulsion copolymerized with an acrylic monomer to provide a product in which the molecules of the dye are part of the polymer chains. U.S. Pat. No. 4,035,316 to Yen discloses the preparation by emulsion polymerization of biocompatible polyacrylate microbeads and attachment of a fluorescent dye thereto through reactive functional groups. In these cases, the particles obtained, although they contain a dye, are limited to a size of about 3 microns or less and are thus substantially smaller than a cell.